1H-NMR-based metabolomic profiling of CSF in early amyotrophic lateral sclerosis.

BACKGROUND: Pathophysiological mechanisms involved in amyotrophic lateral sclerosis (ALS) are complex and none has identified reliable markers useful in routine patient evaluation. The aim of this study was to analyze the CSF of patients with ALS by (1)H NMR (Nuclear Magnetic Resonance) spectroscopy in order to identify biomarkers in the early stages of the disease, and to evaluate the biochemical factors involved in ALS. METHODOLOGY: CSF samples were collected from patients with ALS at the time of diagnosis and from patients without neurodegenerative diseases. One and two-dimensional (1)H NMR analyses were performed and metabolites were quantified by the ERETIC method. We compared the concentrations of CSF metabolites between both groups. Finally, we performed principal component (PCA) and discriminant analyses. PRINCIPAL FINDINGS: Fifty CSF samples from ALS patients and 44 from controls were analyzed. We quantified 17 metabolites including amino-acids, organic acids, and ketone bodies. Quantitative analysis revealed significantly lower acetate concentrations (p = 0.0002) in ALS patients compared to controls. Concentration of acetone trended higher (p = 0.015), and those of pyruvate (p = 0.002) and ascorbate (p = 0.003) were higher in the ALS group. PCA demonstrated that the pattern of analyzed metabolites discriminated between groups. Discriminant analysis using an algorithm of 17 metabolites revealed that patients were accurately classified 81.6% of the time. CONCLUSION/SIGNIFICANCE: CSF screening by NMR spectroscopy could be a useful, simple and low cost tool to improve the early diagnosis of ALS. The results indicate a perturbation of glucose metabolism, and the need to further explore cerebral energetic metabolism.

Proton magnetic resonance spectroscopy to study the metabolic changes in the brain of a patient with Leigh syndrome.

Localized proton magnetic resonance spectroscopy (MRS) was performed to study the metabolic changes in the brain of a patient with Leigh syndrome, who had a T-->G point mutation at nt 8993 of mitochondrial DNA. In this patient, sodium dichloroacetate therapy normalized the lactate and pyruvate levels in both blood and cerebrospinal fluid (CSF). However, his psychomotor retardation did not improve and magnetic resonance imaging showed progressive cerebral atrophy. In the patient's spectra, elevation of brain lactate was observed throughout the brain with regional variations, predominantly in the basal ganglia and brainstem with an abnormal MRI appearance. Although the lactate/creatine ratio observed on proton-MRS was related to the CSF lactate level, the ratio did not completely parallel the CSF lactate level, i.e. brain lactate was detected even when the CSF lactate level had become normalized. Furthermore, proton-MRS revealed a decrease in the N-acetylaspartate/creatine ratio and an increase in the choline/creatine ratio, representing neuronal loss and breakdown of membrane phospholipids. The clinical and MRI findings were well related to the changes in spectroscopically determined brain metabolites. These results indicate that the brain metabolites observed on proton-MRS are useful indicators of a response to therapy and prognosis in Leigh syndrome.

Adrenocorticotropic hormone therapy for infantile spasms alters pyruvate metabolism in the central nervous system.

To clarify the mechanism of action of adrenocorticotropic hormone (ACTH) in treating infantile spasms, we evaluated the effects of ACTH on the metabolism of pyruvate in the central nervous system (CNS) of children with infantile spasms. We measured the levels of lactate and pyruvate in cerebrospinal fluid (CSF) and serum, before and during ACTH treatment in 12 children with infantile spasms. We evaluated statistically any correlation between the observed metabolic changes and the clinical response of ACTH. ACTH therapy significantly elevated the levels of lactate and pyruvate in the CSF and serum. The effect was not dose-dependent. During ACTH therapy, the serum levels of lactate and pyruvate and the ratio of lactate to pyruvate (L:P ratio) were unrelated to these levels in CSF. Patients who showed a good initial response to treatment had a significantly higher CSF level of pyruvate and a lower L:P ratio during therapy than did those with a poor initial response. This is the first report that ACTH therapy administered for infantile spasms alters pyruvate metabolism in the CNS. This metabolic change may be involved in part in the action of ACTH in relieving infantile spasms.

Tardive dyskinesia and substrates of energy metabolism in CSF.

OBJECTIVE: This study was undertaken to assess the relationships among CSF concentrations of substrates of mitochondrial energy metabolism, neuroleptic medication, and neurological side effects. METHOD: CSF was obtained from 25 patients with schizophrenia; seven were unmedicated and 11 had tardive dyskinesia. CSF concentrations of four substrates of mitochondrial energy metabolism (Krebs cycle)--alanine, aspartate, lactate, and pyruvate--were determined. Tardive dyskinesia was measured with the Abnormal Involuntary Movement Scale (AIMS), and parkinsonism was measured with the Simpson-Angus Rating Scale. RESULTS: CSF concentrations of alanine were significantly elevated in the medicated patients when tardive dyskinesia status was controlled for. CSF aspartate concentrations were significantly elevated in patients with tardive dyskinesia when medication status was controlled for and were significantly correlated with total scores on the AIMS. CONCLUSIONS: These results are consistent with a model linking neuroleptic-induced neurological side effects with impairment of mitochondrial energy metabolism, possibly mediated by inhibition of complex 1 of the electron transport chain.

Determination of lactic acid and pyruvic acid in serum and cerebrospinal fluid by ion-exclusion chromatography with a bulk acoustic wave detector.

A chromatographic method base don a combination of ion-exclusion chromatography separation and bulk acoustic wave series piezoelectric quartz crystal detector quantification for the determination of pyruvic acid and lactic acid in serum and cerebrospinal fluid (CSF) was developed. The separation was carried out using a Shim-pak SCR-102H ion-exclusion column with phosphoric acid solution as eluent. The method shows an acceptable detection limit and anti-interference ability. Serum and CSF from healthy individuals and patient were analysed successfully.

Increased cerebrospinal fluid pyruvate levels in Alzheimer's disease.

Impaired energy metabolism is an early, predominant feature in Alzheimer's disease. In order to find out simple, reliable 'in vivo' markers for the clinical-biological typization of the disorder, we measured cerebrospinal fluid (CSF) glucose, lactate and pyruvate levels in patients suffering from dementia of Alzheimer type (DAT) and in healthy elderly controls. DAT group showed remarkably higher levels of pyruvate (P = 0.01), with no overlap with the values obtained in controls. CSF pyruvate levels were also significantly associated with the severity of dementia. Therefore, CSF pyruvate levels neatly separate DAT patients from controls, having also pathogenetic value.

Oxidative metabolism in Rett syndrome: 1. Clinical studies.

The etiology of Rett syndrome (RS) remains a mystery. The clinical phenotype has similarities to that of patients with mitochondrial defects of oxidative metabolism. There is evidence of lactate and pyruvate elevations in blood and CSF in some patients. Over the last 10 years we have studied girls with RS looking for evidence of a defect in oxidative metabolism. We present data on lactate and pyruvate blood measurements in 30 patients with RS with repeated measurements performed over time in many. Taken as a whole the means of measurements of lactate and pyruvate fall within the control range, however, individual patients have marked elevation of both lactate and pyruvate with considerable fluctuation over time. Nine girls with typical RS were studied in detail using a clinical protocol designed to identify disorders of oxidative metabolism. These patients underwent fasting for 24 hours, glucose loading and alanine loading tests. Seven girls had skin and muscle biopsies performed. One patient admitted with particularly high blood lactate levels underwent hourly blood collections over a 24 hour period during which state of alertness was noted and respiratory monitoring was performed. In this patient serial blood sampling for lactate performed. In this patient serial blood sampling for lactate performed with oxypneumocardiogram recording demonstrated a fall in plasma lactate to normal levels during sleep when the respiratory pattern was normal. Such fluctuations of plasma lactate apparently correlated with sleep/wake state and respiration suggest that in some patients with RS lactate elevations may arise from respiratory abnormalities. Other positive findings included prediabetic glucose responses in three girls. Ammonia levels following alanine loading were normal in all patients.

Capillary zone electrophoretic determination of organic acids in cerebrospinal fluid from patients with central nervous system diseases.

Organic acids in cerebrospinal fluid (CSF) from patients with various central nervous system (CNS) diseases were determined by capillary zone electrophoresis (CZE). Under one of the two sets of conditions employed, several anionic components of CSF were separated into corresponding peaks on the electropherograms and determined. The other conditions employed were also useful in measurement of the lactate contents in CSF. The CSF levels of lactate and pyruvate and the ratios of lactate to pyruvate were elevated in patients with cerebral infarction and bacterial meningitis, whereas CSF ascorbate was reduced mainly in inflammatory disorders of the CNS. The results showed that CZE can become a powerful tool in the biochemical diagnosis of CNS diseases.

Critical evaluation of postmortem changes in human autopsy cisternal fluid. Enzymes, electrolytes, acid-base balance, glucose and glycolysis, free amino acids and ammonia. Correlation to total brain ischemia.

By studying early postmortem changes in cerebrospinal fluid (CSF) it is possible to draw conclusions as to premortem focal brain cell injury and terminal brain ischemia. Cisternal fluid (CF) from 40 different adult cadavers with no known neurological disorder was analyzed and compared with known in vivo values. They were divided into four groups (n = 10 in each group), CF samples taken 2, 4, 10, and 24 h after death. The enzyme activity of CK and CK-BB (EC 2.7.3.2) increased linearly and statistically significantly 4-24 h postmortem (P < 0.001) the 2 h values being already 10 to 20 times higher than in vivo, LD and its isoenzymes 1 to 3 (EC 1.1.1.27) distinctly 10 to 24 h after death. Glucose and pyruvate concentrations in the CF declined, as did Na+ and Cl-. Lactate and K+ increased over time. The earliest statistically significant changes between different timepoints were seen in lactate, pyruvate and K+ concentrations. The GABA concentration was already more than 170 times at 2 h postmortem, and glutamate more than 20 times higher than in vivo. The concentrations of alanine, glycine, lysine, histidine, isoleucine, phenylalanine, and tyrosine were 2 to 3 times higher at 2 h postmortem than during life. The concentrations of all amino acids and ammonia increased linearly and statistically significantly (P < 0.001) in the CF 4 to 24 h postmortem.

CSF and serum metabolic profile of patients with Huntington's chorea: a study by high resolution proton NMR spectroscopy and HPLC.

We studied both cerebrospinal fluid (CSF) and serum of 11 patients suffering from Huntington's disease (HD) and 12 control subjects by combining high resolution proton NMR spectroscopy and HPLC. NMR spectroscopy analysis of the CSF shows a significant increase (60%) in pyruvate concentration in HD patients. No unexpected molecules were detected. Glutamate, glutamine, aspartate, proline and GABA levels were found unchanged in the CSF of HD patients, using HPLC analysis. Conversely, a significant increase (30%) in the CSF level of glycine was detected. These observations are in agreement with the metabolic hypothesis of HD physiopathogenesis. In addition, the protocol combining NMR spectroscopy and HPLC provides a straightforward evaluation of brain metabolic status and blood-brain-barrier function.

Pyruvate dehydrogenase deficiency: clinical and biochemical diagnosis.

A female neonate with pyruvate dehydrogenase (PDH) deficiency is presented with clinical, radiologic, biochemical, neuropathologic, and molecular genetic data. She was dysmorphic, with a high forehead, lowset ears, thin upper lip, upturned nose, and rhizomelic limbs. Cranial MRI revealed severe cortical atrophy, ventricular dilatation, and corpus callosum agenesis. Pyruvate and lactate levels were increased in CSF and blood. Urinary organic acid profile was compatible with PDH deficiency. PDH activity was normal in fibroblasts, lymphocytes, and muscle. The PDH E1-alpha gene was sequenced and a single base mutation was found within the regulatory phosphorylation site in exon 10. It is postulated that this mutation causes a cerebral form of PDH deficiency. Tissue-specific expression of the disease could be explained by differential X chromosome inactivation because the PDH E1-alpha gene is located on this chromosome. Dysmorphism with severe cerebral malformations in female patients merits a metabolic evaluation, including determination of lactate and pyruvate levels in CSF.

Long-term monitoring of CSF lactate levels and lactate/pyruvate ratios following subarachnoid haemorrhage.

Ventricular cerebrospinal fluid (CSF) lactate concentrations and lactate/pyruvate (L/P) ratios were measured daily in 20 patients from day 1 to day 12 after subarachnoid haemorrhage due to ruptured aneurysms. Patients without symptomatic vasospasm were classified in Group 1, patients with symptomatic vasospasm were classified in Group 2, and patients who were Hunt and Kosnik grade 4 on admission clinically were classified in Group 3. Patients in all three groups had high CSF lactate concentrations on day 1, and, especially in Group 3, the high lactate was accompanied by an increased L/P ratio and a decreased CSF bicarbonate. Lactate concentrations in Group 1 decreased throughout the observation period. Lactate concentrations in Group 2 also decreased but then began to increase again on days 5 to 7, correlating well with the onset of cerebral vasospasm. The delayed increase of CSF lactate in Group 2 was also accompanied by increases in the CSF pyruvate level and the CSF L/P ratio. Daily monitoring of CSF lactate may thus serve as a chemical marker for cerebral vasospasm.

Is Parkinson's disease a mitochondrial disorder?

Parkinson's disease (PD) is a common degenerative disease, but its etiology is still unknown. However, since the discovery of MPTP, many investigators have been interested in the mitochondrial function in PD. We investigated mitochondrial functions in PD patients using the methods which have successfully been applied to mitochondrial myopathies (MM), i.e. assay of lactate and pyruvate, measurement of muscle mitochondrial respiratory enzyme activities and Southern blot analysis of muscle mitochondrial DNA. Parkinson's disease patients did not differ from controls in the mean blood and CSF (cerebrospinal fluid) lactate and pyruvate levels at the basal resting state or during an aerobic exercise. But mitochondrial complex I activity of the skeletal muscle was significantly decreased in PD. In the Southern blot analysis, we could not find major deletions or insertions of mitochondrial DNA in PD. Our studies disclosed a differential mitochondrial impairment between PD and MM. We discuss the implication of our observation.

The Rett syndrome and CSF lactic acid patterns.

We investigated both blood and cerebrospinal fluid (CSF) lactate and pyruvate levels in seven girls with the Rett syndrome (RS) and evaluated the relationship between CSF lactate and pyruvate levels and the clinical manifestations, particularly seizures, anticonvulsant medication, and breathing dysfunction including breath holding, apnea and hyperventilation. Elevated lactate and pyruvate levels in CSF with normal serum lactate were found in two RS patients. Elevated CSF lactate correlated significantly with the clinical occurrence of hyperventilation (P0 = 0.048, Fisher exact probability). We measured native and dichloroacetate (DCA)-activated pyruvate dehydrogenase (PDH) complex activities in two patients (#1 and 2) using cultured lymphoblastoid cell lines which were transformed by EB virus and the results were normal. We also analyzed CSF citric acid intermediates from 7 RS patients including citric acid, cis-aconitate, alpha-ketoglutarate, succinate, fumarate, malate and oxaloacetate. These concentrations were not significantly different from those control patients (N = 21). An elevated lactate level may be a clue to clarify the etiology of RS.

Marked reduction in CSF lactate and pyruvate levels after CoQ therapy in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS).

Many CoQ trials for mitochondrial encephalomyopathy are reported, however, the action of CoQ in the central nervous system is unknown. We administered CoQ to a patient with MELAS, and decreasing CSF lactate and pyruvate levels were revealed. This reduction in CSF lactate and pyruvate may be evidence that CoQ acts directly on the CNS. There have been no other descriptions of evidence of CoQ effective action in the central nervous system, a finding unique to this report.

MELAS syndrome. Report of two patients, and comparison with data of 24 patients derived from the literature.

We present two unrelated MELAS patients, and compare them with 24 patients derived from the literature. In most patients the stroke-like features of the MELAS syndrome occur late in the course of the disease. The diagnosis is based on characteristic clinical symptoms, presence of lactic acidemia, mitochondriopathy in muscle, and low density lesions on cerebral CT, most frequently occurring in the posterior and parieto-temporal regions. In some cases, a metabolic defect could not be demonstrated, in other cases a partial deficiency of various respiratory chain enzymes was found.

Interstitial and cerebrospinal fluid levels of energy-related metabolites after middle cerebral artery occlusion in rats.

This investigation was designed to study the dynamics of energy-related metabolites (i.e., lactate, pyruvate, inosine, and hypoxanthine) in the extracellular fluid (ECF) of the striatum and in cisternal cerebrospinal fluid (CSF) during the first 6 h after middle cerebral artery occlusion (MCAO) using microdialysis. Ischemia induced a dramatic increase in the ECF levels of lactate, inosine, and hypoxanthine, while pyruvate did not change significantly. The major part of these changes occurred during the first 10 min after MCAO. Inosine tended to normalize towards the end, while lactate and hypoxanthine remained elevated throughout the experiment. There was no increase of the energy-related metabolites in CSF during the experiment. It was concluded that lactate, inosine, and hypoxanthine appear to be useful ECF markers of the compromised energy state of the brain during ischemia. Because the metabolites did not appear in CSF during the first 6 h after MCAO, such measurements seem not to be useful for early detection of a disturbance in energy metabolism.